Nitro-substituted non-fluorescent asymmetric cyanine dye compounds

ABSTRACT

The invention provides an asymmetric cyanine dye compound having the structure                    
     including substituted forms thereof, wherein, at least one of R 1  and R 2  is linking group, X is O, S, or Se, and n ranges from 0 to 2. The invention further provides reporter-quencher dye pairs comprising the asymmetric cyanine dyes, dye-labelled polynucleotides incorporating the asymmetric cyanine dyes, and hybridization detection methods utilizing the dye-labelled polynucleotides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/602,544, filed Jun. 21, 2000, now U.S. Pat. No. 6,541,618 which is adivisional of U.S. application Ser. No. 09/012,525, filed Jan. 23, 1998,now U.S. Pat. No. 6,080,868, both of which are incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to dye compounds useful as quenchers in areporter-quencher energy-transfer dye pair. More specifically, thisinvention relates to cyanine quencher compounds, reagents incorporatingsuch compounds and methods utilizing such compounds and/or reagents.

BACKGROUND

Nucleic acid hybridization assays comprise an important class oftechniques in modern biology. Such assays have diverse applicationsincluding the diagnosis of inherited disease, human identification,identification of microorganisms, paternity testing, virology, and DNAsequencing, i.e., sequencing by hybridization.

An important aspect of nucleic acid hybridization assays is the methodused to facilitate detection of the hybridization event. A particularlyimportant class of methods used in nucleic acid hybridization assaysemploys a reporter-quencher energy-transfer dye pair comprising a“reporter” dye and a “quencher” dye which interact through afluorescence resonance energy transfer (FRET) process. In these methods,the reporter is a luminescent compound that can be excited either bychemical reaction, producing chemiluminescence, or by light absorption,producing fluorescence. The quencher can interact with the reporter toalter its light emission, usually resulting in the decreased emissionefficiency of the reporter. This phenomenon is called quenching. Theefficiency of quenching is a strong function of the distance between thereporter molecule and the quencher molecule. Thus, in a nucleic acidhybridization assay, detection of a hybridization event is accomplishedby designing an energy transfer system in which the spacing between areporter and a quencher is modulated as a result of the hybridization.

Quenchers which are presently used in FRET-based nucleic acidhybridization assays are themselves fluorescent. That is, in addition toquenching the fluorescence of the reporter, the quencher producesfluorescent emissions. This is problematic, particularly in assaysemploying multiple spectrally-resolvable reporters, because the quencherfluorescence can interfere with the fluorescent signal produced by oneor more of the reporters.

Thus, there remains a continuing need for quencher dyes which arethemselves substantially non-fluorescent.

SUMMARY

The present invention is directed towards our discovery of a class ofnon-fluorescent cyanine quencher compounds which are useful in thecontext of a reporter-quencher energy-transfer dye pair. These quenchercompounds find particular application in nucleic acid hybridizationassays employing fluorescence energy transfer as a means of detection.

In a first aspect, the invention comprises an asymmetric cyanine dyecompound having the structure

including substituted forms thereof, wherein at least one of R₁ and R₂is linking group, X is O, S, or Se, and n ranges from 0 to 2.

In a second aspect, the invention includes a reporter-quencherenergy-transfer dye pair comprising a reporter dye and a quencher dye,wherein the quencher dye is an asymmetric cyanine dye compound of thefirst aspect.

In a third aspect, the invention includes a an oligonucleotide having acyanine dye quencher according to the first aspect covalently attachedthereto.

In a fourth aspect, the invention provides a method for detecting atarget nucleic acid sequence including the steps of providing a samplenucleic acid including at least one target nucleic acid sequence, andhybridizing a labelled oligonucleotide probe to the target nucleic acidsequence, the labelled oligonucleotide probe being labelled with anasymmetric cyanine dye compound of the first aspect. In a particularlypreferred embodiment of this fourth aspect, the method further includesthe step of digesting the oligonucleotide probe such that one or both ofthe reporter and quencher dyes is removed from the oligonucleotideprobe.

Various aspects and embodiments of the above-described invention achieveone or more of the following important advantages over known quencherdye compounds. The asymmetric cyanine quenchers of the present inventionmay be easily covalently linked to a reagent, e.g., a polynucleotide.Furthermore, oligonucleotide probes labelled with the asymmetric cyaninequenchers of the present invention exhibit enhanced hybridizationstability as compared to conventionally labelled probes, therebyallowing for the use of shorter probes which are more sensitive tohybridization mismatches. In addition, the asymmetric cyanine quenchersof the present invention are essentially non-fluorescent, therebyproviding additional spectrum which can be occupied by one or moreadditional reporters.

These and other objects, features, and advantages of the presentinvention will become better understood with reference to the followingdescription, drawings, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show several preferred cyanine dye compounds of thepresent invention.

FIGS. 2A and 2B show a generalized synthetic scheme for the synthesis ofthe cyanine dye quenchers of the present invention.

FIGS. 3A-3C show a synthetic scheme for the synthesis of a firstpreferred cyanine dye quencher of the present invention.

FIGS. 4A and 4B show a synthetic scheme for the synthesis of a secondpreferred cyanine dye quencher of the present invention.

FIGS. 5A-5E shows a schematic depiction of several hybridizationdetection methods according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

I. Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Energy transfer” and “fluorescence quenching” refer to a processeswhereby energy is removed from an electronically excited luminescent“reporter” molecule by a “quencher” molecule, thereby returning thereporter molecule to its ground state without the emission of light fromthe reporter molecule. The reporter molecule may be excited to one ofits higher energy levels by any of a number of process, including lightabsorption and chemical reaction.

“Spectral resolution” in reference to a set of dyes means that thefluorescent emission bands of the dyes are sufficiently distinct, i.e.,sufficiently non-overlapping, that reagents to which the respective dyesare attached, e.g. polynucleotides, can be distinguished on the basis ofa fluorescent signal generated by the respective dyes using standardphotodetection systems, e.g. employing a system of band pass filters andphotomultiplier tubes, charged-coupled devices and spectrographs, or thelike, as exemplified by the systems described in U.S. Pat. Nos.4,230,558, 4,811,218, or in Wheeless et al, pgs. 21-76, in FlowCytometry: Instrumentation and Data Analysis (Academic Press, New York,1985).

“Linking group” means a moiety capable of reacting with a “complementaryfunctionality” attached to a reagent, such reaction forming a “linkage”connecting a dye to a reagent. Preferred linking groups includeisothiocyanate, sulfonyl chloride, 4,6-dichlorotriazinyl, carboxylate,succinimidyl ester, or other active carboxylate whenever thecomplementary functionality is amine. Alternatively, the linking groupmay be amine. Preferably the linking group is maleimide, halo acetyl, oriodoacetamide whenever the complementary functionality is sulfhydryl.See R. Haugland, Molecular Probes Handbook of Fluorescent Probes andResearch Chemicals, Molecular probes, Inc. (1992). In a particularlypreferred embodiment, the linking group is a N-hydroxysuccinimidyl (NHS)ester and the complementary functionality is an amine, where to form theNHS ester, a dye of the invention including a carboxylate linking groupis reacted with dicyclohexylcarbodiimide and N-hydroxysuccinimide.

“Lower alkyl” denotes straight-chain and branched hydrocarbon moietiescontaining from 1 to 8 carbon atoms, e.g., methyl, ethyl, propyl,isopropyl, tert-butyl, isobutyl, sec-butyl, neopentyl, tert-pentyl, andthe like.

“Nucleoside” refers to a compound consisting of a purine, deazapurine,or pyrimidine nucleoside base, e.g., adenine, guanine, cytosine, uracil,thymine, deazaadenine, deazaguanosine, and the like, linked to a pentoseat the 1′ position. When the nucleoside base is purine or 7-deazapurine,the sugar moiety is attached at the 9-position of the purine ordeazapurine, and when the nucleoside base is pyrimidine, the sugarmoiety is attached at the 1-position of the pyrimidine, e.g., Kombergand Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992). Theterm “nucleotide” as used herein refers to a phosphate ester of anucleoside, e.g., triphosphate esters, wherein the most common site ofesterification is the hydroxyl group attached to the C-5 position of thepentose. The term “nucleoside/tide” as used herein refers to a set ofcompounds including both nucleosides and nucleotides. “Analogs” inreference to nucleosides/tides include synthetic analogs having modifiedbase moieties, modified sugar moieties and/or modified phosphatemoieties, e.g. described generally by Scheit, Nucleotide Analogs (JohnWiley, New York, 1980). Phosphate analogs comprise analogs of phosphatewherein the phosphorous atom is in the ⁺5 oxidation state and one ormore of the oxygen atoms is replaced with a non-oxygen moiety. Exemplaryphosphate analogs include but are not limited to phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate,boronophosphates, including associated counterions, e.g., H⁺, NH₄ ⁺,Na⁺, if such counterions are present. Exemplary base analogs include butare not limited to 2,6-diaminopurine, hypoxanthine, pseudouridine,C-5-propyne, isocytosine, isoguanine, 2-thiopyrimidine, and other likeanalogs. Exemplary sugar analogs include but are not limited to 2′- or3′-modifications where the 2′- or 3′-position is hydrogen, hydroxy,alkoxy, e.g., methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxyand phenoxy, amino or alkylamino, fluoro, chloro and bromo. The term“labelled nucleoside/tide” refers to nucleosides/tides which arecovalently attached to a label.

“Polynucleotide” or “oligonucleotide” means polymers of naturalnucleotide monomers or analogs thereof, including double and singlestranded deoxyribonucleotides, ribonucleotides, α-anomeric formsthereof, and the like. Usually the nucleoside monomers are linked byphosphodiester linkages, where as used herein, the term “phosphodiesterlinkage” refers to phosphodiester bonds or bonds including phosphateanalogs thereof, including associated counterions, e.g., H⁺, N₄ ⁺, Na⁺,if such counterions are present. Polynucleotides typically range in sizefrom a few monomeric units, e.g. 5-40, to several thousands of monomericunits. Whenever a polynucleotide is represented by a sequence ofletters, such as “ATGCCTG,” it will be understood that the nucleotidesare in 5′→3′ order from left to right and that “A” denotesdeoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine,and “T” denotes deoxythymidine, unless otherwise noted.

“Substituted” as used herein refers to a molecule wherein one or morehydrogen atoms are replaced with one or more non-hydrogen atoms,functional groups or moieties. For example, an unsubstituted nitrogen is—NH₂, while a substituted nitrogen is —NHCH₃. Exemplary substituentsinclude but are not limited to halo, e.g., fluorine and chlorine, loweralkyl, lower alkene, lower alkyne, sulfate, sulfonate, sulfone, amino,ammonium, amido, nitrile, lower alkoxy, phenoxy, aromatic, phenyl,polycyclic aromatic, electron-rich heterocycle, and linking group.

“Methine bridge” or “polymethine bridge” refers to a portion of acyanine dye compound connecting two base portions, the bridge having thefollowing structure

—(CH═CH)n-CH—,

where n typically ranges from 0 to 2.

“Xanthene dyes” are dyes which comprise the following fused three-ringstructure

where R is oxygen (fluorescein) or —NH (rhodamines), includingsubstituted forms thereof. Exemplary substituted fluorescein compoundsinclude the “NED” dye which has the structure

the “TET” dye which has the structure

and the “FAM” dye which has the structure

An exemplary rhodamine dye is the “TAMRA” or “TMR” dye which has thestructure

“Coumarin dyes” are dyes which comprise the following fused two-ringstructure

where R is oxygen (hydroxycoumarin) or —NH (aminocoumarin), includingsubstituted forms thereof.

“BODIPY™ dyes” are dyes comprising the following fused ring structure

including substituted forms thereof. See the Handbook of FluorescentProbes and Research Chemicals, Sixth Addition, Haugland, MolecularProbes, Inc. (1996).

“Cyanine dyes” are dyes comprising two nitrogen-heterocyclic ringsjoined by a methine, or polymethine, bridge. An exhaustive review ofsuch dyes is provided by Ficken (Ficken, The Chemistry of SyntheticDyes, Vol IV, Venkataraman (1971)).

I. Asymmetric Cyanine Dye Compounds

A. Structure

In a first aspect, the present invention comprises a novel class ofcyanine dye compounds useful as non-fluorescent quenchers. Thesecompounds have the general structure shown in Formula 1 immediatelybelow, including substituted forms thereof, where at least one of R₁ andR₂ is linking group, X is O, S, or Se, and n ranges from 0 to 2. (Notethat all molecular structures provided herein are intended to encompassnot only the exact electronic structures presented, but also include allresonant structures, protonation states and associated counterionsthereof)

Preferably, when the compound is to be used as a non-fluorescentquencher, the compound includes a nitro C-3 substituent. For example,compounds 5, 9, 20, 21 and 23.

In an alternative preferred embodiment, the linking group of thecompound of Formula 1 is a lower alkylamine or a lower alkylcarboxymoiety, where a particularly preferred lower alkylcarboxy is—(CH₂)_(n)N⁺(CH₃)₂(CH₂)_(n)CO₂H, where n ranges from 2 to 12. Forexample, compound 23.

In another preferred embodiment, one of R₁ or R₂ is —(CH₂)_(n)N^(+ (CH)₃)₃, where n ranges from 2 to 12, and the other of R₁ or R₂ is linkinggroup.

In yet another preferred embodiment, the X-group in the cyaninecompounds of the invention is sulfur. For example, compounds 5, 9, 20,21, 22 and 23.

In another preferred embodiment, the compound of Formula 1 includes afused aromatic, or substituted aromatic, substituent bonded at positions1 and 2, positions 2 and 3; and/or positions 3 and 4. More preferably,the substituted aromatic includes one or more nitro substituents. Forexample, compounds 21 and 22.

In an additional preferred embodiment of the present invention, thecompound of Formula 1 includes a bridging group that when taken togetherwith R₁ and the proximate carbon of the methine bridge forms a ringstructure having 5 to 7 members, more preferably 6 members. For example,compounds 20 and 21.

B. Synthesis

Generally, the non-fluorescent cyanine dye quenchers may be prepared asfollows. See FIGS. 2A and 2B. A quaternized benzazole derivative, e.g. abenzothiazolium salt 10 or 13, is mixed with a lepidinium salt, 11, andrefluxed under basic conditions, e.g. diisopropylethylamine in methanol,or pyridine. The solvent is evaporated and the crude solid is washedwith dilute hydrochloric acid, e.g., 5% in water, and dried.

The dyes may be rendered amino-reactive by converting a carboxylic acidgroup to a succinimidyl ester. For example, dye 12 or 14 is dissolved inDMF with succinimidyl tetramethyluronium salt and DIPEA. The product isprecipitated by the addition of dilute HCl, washed and dried.

II. Dye Pairs Including Non-Fluorescent Cyanine Dyes

Reporter-quencher dye pairs may be composed of any pair of moleculeswhich can participate in an energy transfer process. Exemplary reportersmay be selected from xanthene dyes, including fluoresceins, andrhodamine dyes. Many suitable forms of these compounds are commerciallyavailable with various substituents on their xanthene rings which can beused as the site for bonding or as the bonding functionality forattachment to an oligonucleotide. Another group of fluorescent compoundsare the naphthylamines, having an amino group in the alpha or betaposition. Included among such naphthylamino compounds are1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonateand 2-p-touidinyl-6-naphthalene sulfonate. Other dyes include but arenot limited to 3-phenyl-7-isocyanatocoumarin, acridines, such as9-isothiocyanatoacridine and acridine orange,N-(p-(2-benzoxazolyl)phenyl)maleimide, benzoxadiazoles, stilbenes,pyrenes, and the like.

Preferably, reporter molecules are selected from fluorescein andrhodamine dyes. These dyes and appropriate linking methodologies forattachment to oligonucleotides are described elsewhere (Khanna et al(cited above); Marshall, Histochemical J., 7: 299-303 (1975); Menchen etal, U.S. Pat. No. 5,188,934; Menchen et al, European Patent ApplicationNo. 87310256.0; and Bergot et al, International ApplicationPCT/US90/05565). Particularly preferred reporter molecules includefluorescein dyes NED, TET and FAM.

Exemplary reporter-quencher pairs include the following:

Reporter Quencher FAM Nitrothiazole Orange (Compound 9) FAMNitrothiazole Blue (Compound 5) TET Nitrothiazole Blue (Compound 5) TETNitrothiazole Blue (Compound 5) NED Nitrothiazole Blue (Compound 5)

III. Dye-Labelled Polynucleotides

In another aspect, the present invention comprises polynucleotideslabelled with the non-fluorescent cyanine dyes of the invention. Suchlabelled polynucleotides are useful in a number of important contextsincluding as oligonucleotide hybridization probes and oligonucleotideligation probes.

Singly- or doubly-labelled polynucleotides may be prepared using any ofa number of well known methods. Methods suitable for labeling anoligonucleotide at the 3′ end include but are by no means limited to (1)periodate oxidation of a 3′-terminal ribonucleotide, followed byreaction with an amine-containing label (Heller and Morrison, In RapidDetection and Identification of Infectious Agents (D. T. Kingsbury andS. Falkow, eds.), pp 245-256, Academic Press (1985)); (2) enzymaticaddition of a 3′-aliphatic amine-containing nucleotide usingdeoxynucleotidyl transferase, followed by reaction with anamine-reactive label (Morrison, European Patent Application No. 232967); and (3) periodate oxidation of a 3′-ribonucleotide, followed byreaction with 1,6-hexanediamine to provide a 3′-terminal aliphaticamine, followed by reaction with an amine-reactive label (Cardullo etal., Proc. Natl. Acad. Sci. USA, 85: 8790-8794 (1988)).

Methods for labeling the 5′ end of an oligonucleotide include (1)periodate oxidation of a 5′-to-5′-coupled ribonucleotide, followed byreaction with an amine-reactive label (Heller and Morrison, 1985); (2)condensation of ethylenediamine with 5′-phosphorylatedpolynucleotide,followed by reaction with an amine reactive label (Morrison, 1987); and(3) introduction of an aliphatic amine substituent using an aminohexylphosphite reagent in solid-phase DNA synthesis, followed by reactionwith an amine reactive label (Cardullo, 1988).

In addition to these end-labeling methods, labels can be placed atspecific locations within synthetic polynucleotides usingamine-containing nucleotide phosphoramidite reagents, e.g.,5′-dimethoxytrityl-5-[N-trifluoroacetylaminohexyl)-3-acrylimido]-2-deoxyuridine,3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, e.g.,Amino-Modifier C6 dT phosphoramidite (Linker Arm Nucleotide, GlenResearch, Inc.) (Mathies et al., U.S. Pat. No. 5,688,648).

For a through review of oligonucleotide labeling procedures see R.Haugland in Excited States of Biopolymers, Steiner ed., Plenum Press(1983), Fluorogenic Probe Design and Synthesis: A Technical Guide, PEApplied Biosystems (1996), and G. T. Herman, Bioconjugate Techniques,Academic Press (1996).

Generally, the design of oligonucleotide hybridization probes includingthe non-fluorescent cyanine dye quenchers of the invention followsconventional teachings. Thus, in designing labelled oligonucleotidehybridization probes, the following general guidelines should befollowed: (1) if the target nucleic acid sequence is located within aPCR amplicon, the probe sequence should be such that the probehybridizes at a location on the sequence between the PCR primers; (2)probes should be about 20-30 nucleotides long so as to ensure goodhybridization kinetics and specificity of binding; (3) avoid secondarystructure in the probe and target nucleic acid sequence; (4) if theprobe is being used in combination with a pair of PCR primers, the probeshould not hybridize to either of the forward and reverse primers; and(5) avoid probes with long stretches of a single nucleotide, i.e., morethan four; and (6) when choosing between a probe sequence and itscomplement, pick the strand that has more C nucleotides than Gnucleotides.

IV. Hybridization Methods Utilizing Non-Fluorescent Cyanine Dyes

Several hybridization assay formats that employ energy transfer as ameans for detecting hybridization have been described, five of which arediscussed below and shown schematically in FIGS. 5A-E.

In a first assay format, shown in FIG. 5A, the sequences of twooligonucleotide probes are selected such that they will hybridize tocontiguous regions of a target nucleic acid 5. The first probe 10,hybridizing toward the 5′-terminus of the target nucleic acid, islabelled near its 5′-terminus with a reporter label, whereas the secondprobe 15 is labelled near its 3′-terminus with a quencher label. Thus,when a 3-way hybrid 20 is formed among the target nucleic acid 5 and thefirst 10 and second 15 probes, the reporter and quencher are broughtinto close proximity and energy transfer can take place. Thus, in thisformat, the emission of the reporter is quenched upon the hybridizationof the two probes to the target. (Heller et al., European PatentApplication No. 070 685 (1983))

In a second assay format, shown in FIG. 5B, two oligonucleotide probes25 and 30 which are complementary to each other and which each contain areporter or a quencher label are used. The location of the labels isselected such that when the probes are hybridized to one another to forma probe-probe hybrid 35, the quenching interaction is favored, whereasan insignificant amount of quenching occurs when the probes areseparated. The detection of target nucleic acid is achieved bydenaturing both the target nucleic acid 5 and the probes 25 and 30, andthen allowing the strands to reanneal. Thus, there is a competitionbetween probe-probe hybridization and probe-target hybridization. Themore target nucleic acid that is present, the larger the number ofprobes that will hybridize to the target forming probe-target hybrids40. The presence of target DNA is indicated by an increased emissionfrom the reporter R due to the reduced quenching by the quencher Qcaused by a reduction in the number of probe-probe hybrids. (Morrison,European Patent Application 232 967 (1987)).

A third assay format, depicted in FIG. 5C, uses only one labelled probe45 and a dye that binds preferentially to double-stranded nucleic acid50. This dye 50 may intercalate between the base pairs of thedouble-stranded species or may bind to the outside of the helix andserves as a quencher. Thus, in the absence of hybridization, thequencher Q does not bind to the single-stranded probe 45, and thereporter R is unaffected by Q. However, in the presence of a targetnucleic acid 5, the probe 45 hybridizes to the target nucleic acid and Qbinds to the resulting double-stranded region forming a target-probe-dyecomplex 55. In the complex, Q and R are placed in close proximity andenergy transfer or fluorescence quenching may take place. Thus, in thisformat, the emission of the reporter is quenched upon the hybridizationof the probe to the target. (Heller and Morrison, In Rapid Detection andIdentification of Infectious Agents (D. T. Kingsbury and S. Falkow,eds.), pp 245-256, Academic Press (1985)).

In a fourth assay format, shown in FIG. 5D, a single probe 60 is usedwhich is labelled with both a reporter and a quencher. The location ofthe reporter and quencher labels is selected so that when the probe isin a single stranded state, i.e., unhybridized to a target nucleic acid,the single-stranded conformation of the probe is such that the reporterand quencher labels are in close proximity thereby allowing energytransfer to take place. In one alternative method of achieving thissingle-stranded confirmation, the reporter and quencher are brought intoclose proximity by designing the probe sequence such that a hairpinforms at the ends of the probe thereby forcing the reporter and quenchertogether (Bagwell, European patent Application No. 601 889 (1994); Tyagiand Kramer, Nature Biotechnology, 14: 303-308 (1996)). In anotheralternative method for achieving this single-stranded conformation, thereporter and quencher are located far enough apart on the probe suchthat the random-coil confirmation of the single-stranded probe serves tobring the quencher and reporter into sufficiently close proximity(Mayrand, U.S. Pat. No. 5,691,146). However, when the double-labelledprobe 60 is hybridized to a target nucleic acid 5 forming a probe-targethybrid 65, the reporter and quencher are separated from one another, andthe quenching interaction is prevented. Thus, in this format, theemission of the reporter becomes unquenched upon the hybridization ofthe probe to the target.

In a fifth assay format, referred to herein as the “Taqman” assay andillustrated in FIG. 5E, a doubly-labelled probe including both areporter label and a quencher label is digested upon hybridization to atarget nucleic acid thereby liberating one or both of the labels fromthe probe (Holland et al., Proc. Natl. Acad. Sci. USA, 88: 7276-7280(1991); Livak, U.S. Pat. No. 5,538,848). In this method thedoubly-labelled probe 75 is hybridized to a target nucleic acid 5. Inaddition, an oligonuclectide primer 70 is hybridized to the targetnucleic acid at a position upstream from the probe, i.e., closer to the3′-end of the target nucleic acid. The primer 70 is than extend using apolymerase enzyme thereby forming an extended primer 80, e.g., using aDNA polymerase. During the primer extension reaction, a 5′-3′ nucleaseactivity of the polymerase serves to cut the probe 75 so as to form afirst probe fragment 85 including the reporter label and a second probefragment 90 including the quencher label. Thus, the reporter andquencher labels are separated thereby preventing energy transfer betweenthe two. Thus, in this format, the emission of the reporter becomesunquenched upon the hybridization of the probe to the target andsubsequent digestion of the probe.

Note that in each of the five assay formats discussed above and depictedin FIGS. 5A-E, unless otherwise specified, the location of the reporterand quencher is arbitrary. That is, while the reporter may be depictedon one probe and the quencher on another probe, their positions may bereversed.

While the assay formats described above are represented in terms ofsystems employing only a single reporter label, multi-reporter systemsmay also be practiced. Such multi-reporter systems are advantageous inapplications requiring the analysis of multiple hybridization events ina single reaction volume. In such systems, each of the reportermolecules produce emissions which are spectrally resolvable from theemissions from any of the other reporters. The particular quencher usedwith each reporter can be the same or different, depending on thespectral properties of the quencher and reporter.

Each of the assays described above may be conducted in combination witha nucleic acid amplification step, e.g., PCR. That is, prior toconducting the hybridization assay, all or part of the nucleic acidsample may be amplified. When performed in combination with anamplification step, the hybridization-assay may be conducted in anend-point mode or a real-time mode. In an end-point mode, thehybridization assay is performed after the amplification reaction iscomplete, e.g., after all or substantially all of the amplificationcycles of a PCR reaction have been completed. In a real-time mode, ahybridization assay is performed multiple times during the amplificationreaction, e.g., after each thermocycle of a PCR process (Higuchi,European Patent Application No. 512 334). The real-time mode ispreferred when a quantitative measure of the initial amount of targetnucleic acid is required, e.g., the copy-number of pathogen nucleic acidpresent in a blood sample.

EXAMPLES

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention and not to in any way limit its scope.

Example 1 Synthesis of Nitrothiazole Blue 5 and Nitrothiazole Orange 9

The syntheses of nitrothiazole blue 5 and nitrothiazole orange 9 areoutlined in FIGS. 3 and 4.

Preparation of 6-nitrobenzothiazole 1

See FIG. 3A. Nitration of 2-methylbenzothiazole was performed followingthe method of Mizuno, J. Pharm. Soc. Japan, 72, 745 (1952). A mixture offuming nitric acid (1.6 mL) and concentrated sulfuric acid (1.2 mL) wasadded to an ice-cooled solution of 2-methylbenzothiazole (2 g) insulfuric acid (8 mL). The solution was allowed to warm to roomtemperature for one hour, then poured onto 100 mL of ice. The solid wasfiltered, washed with water, and recrystallized from ethanol (80 mL) toprovide 2.5 g of yellowish needles.

Preparation of 3-methyl-6-nitrobenzothiazolium p-toluenesulfonate 2

See FIG. 3A. A mixture of 6-nitrobenzothiazole (1 g) andmethyl-p-toluenesulfonate (1.2 g) was heated to 140° C. for 20 min. Thesolid was washed with acetone and filtered to provide a bluish solid(1.1 g).

Preparation of 2-(2′-acetanilidovinyl)-3-methylbenzothiazoliump-toluenesulfonate 3

See FIG. 3A. A mixture of 3-methyl-6-nitrobenzothiazoliump-toluenesulfonate 2 (200 mg, 0.52 mmol), diphenylformamidine (160 mg,0.8 mmol) and acetic anhydride (2 mL) was refluxed for 20 min. Thecooled solution was triturated with ether to provide a dark brown solid(200 mg).

Preparation of 1-(5′-carboxypentyl)-lepidinium bromide 4

See FIG. 3B. A mixture of lepidine (5 g) and 6-bromohexanoic acid (10 g)was heated to 130° C. for 6 h. The solid was washed with acetone andfiltered to provide an off-white solid (10.5 g).

Preparation of Nitrothiazole Blue 5

See FIG. 3C. A mixture of the acetanilide 3 (65 mg, 0.13 mmol) and thelepidinium bromide 4 (66 mg, 0.2 mmol) and pyridine (1 mL) was combinedand refluxed for 30 min. The blue solution was concentrated to drynessand washed with 5×1 mL 5% HCl. The residue was dried to provide a bluesolid (67 mg).

Preparation of Nitrothiazole Blue Succinimidyl Ester

To a solution of nitrothiazole blue 5 (31 mg, 0.056 mmol) indimethylformamide (0.5 mL) and diisopropylethylamine (0.05 mL) was addedO-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (34mg, 0.12 mmol). The mixture was warmed to 70° C. for 10 min. Reactionprogress was monitored by TLC on silica gel using 600:60:16dichloromethane:methanol:acetic acid as the eluant. To the homogeneoussolution was added 5% HCl (2 mL). The precipitate was washed withadditional HCl and dried to provide a dark solid (30 mg).

Preparation of 2-(methylthio)-6-nitrobenzothiazole 7

See FIG. 4A. Fuming nitric acid (1.93 g) was added dropwise to asolution of 2-(methylthio)benzothiazole (5 g) in concentrated sulfuricacid (16.8 g) cooled in an ice bath. After stirring at 5° C. for 3 h thesolution was poured onto ice and filtered to provide a yellow solid (5.7g, 25 mmol, 91%).

Preparation of 3-methyl-2-(methylthio)-benzothiazoliump-toluenesulfonate 8

A mixture of 6-nitro-2-(methylthio)benzothiazole 7 (0.5 g, 2.2 mmol) andmethyl-p-toluenesulfonate (3.7 g, 20 mmol) was heated from 120° C. to145° C. over one hour. To the cooled solution was added 30 mL of ether.The resulting amorphous solid was triturated with acetone to provide apale mauve solid (0.57 g, 1.4 mmol, 63%).

Preparation of Nitrothiazole Orange 9

See FIG. 4B. To a solution of 3-methyl-2-(methylthio)-benzothiazoliump-toluenesulfonate 8 (50 mg, 0.12 mmol) and1-(5′-carboxypentyl)-lepidinium bromide 4 (41 mg, 0.12 mmol) in methanol(5 mL) was added diisopropylethylamine (0.2 mL). The solution wasrefluxed for 15 min. The solvent was evaporated and the reaction residuetriturated with 5% HCl (2 mL). The solid was washed with additional 5%HCl and dried to provide an orange solid (8 mg).

Preparation of Nitrothiazole Orange Succinimidyl Ester 10

To a solution of nitrothiazole orange 9 (8 mg) in dimethylformamide (0.1mL) and diisopropylethyl amine (0.01 mL) was addedO-(N-succinimidyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (10mg). The mixture was warmed to 70° C. for 10 min. Reaction progress wasmonitored by TLC on silica gel using 600:60:16dichloromethane:methanol:acetic acid as the eluant. To the homogeneoussolution was added 5% HCI (1 mL). The precipitate was washed withadditional HCl and dried to provide an orange solid (8 mg).

Example 2 Preparation of Doubly-Labelled Probe for Taqman Assay

Automated synthesis of oligonucleotide probes was performed using anApplied Biosystems Model 394 DNA/RNA synthesizer (The Perkin-ElmerCorporation, PE Applied Biosystems Division (ABD)) according to thegeneral procedures described in the operators manual. Theoligonucleotides were synthesized in 0.2 μmol scale using dye-labelledCPG solid supports (Mullah and Andrus, Tetrahedron Letters, 38(33):5751-5754 (1997)), DNA FastPhosphoramidites (User Bulletin number 85,1994, ABD) and dye-labelled phosphoramidites, FAM and TET (User Bulletinnumber 78, 1994, ABD). The standard 0.2 μmol synthesis cycle wasslightly modified by extending coupling time of FAM amidite by anadditional 120 sec (User Bulletin number 78, 1994, ABD). Each probeincluded a reporter dye attached to a 5′-end of and a quencher dyelocated at a 3′-end of the probe.

After completion of the synthesis, oligonucleotides were autocleavedfrom the support on the DNA synthesizer by treating with a mixture ofMeOH:t-BuNH₂:H₂O (1:1:2) (Woo et al., U.S. Pat. No. 5,231,191) using a 1hr autocleavage procedure (“END CE” procedure) as described in theoperators manual for the Applied Biosystems Model 394 DNA/RNAsynthesizer. Base protecting groups were removed by heating the mixtureat 85° C. for 1 hr or at 65° C. for 3 h.

The crude oligonucleotides were analyzed for purity and integrity byreverse phase HPLC using the following equipment and conditions: PerkinElmer Series 200 solvent delivery system equipped with ABI 783Aprogrammable detector; Perkin Elmer ISS200 autosampler; and PE Nelson900 series data system; RP-18 reverse phase chromatography column(220×4.6 mm, ABD); solvent A: 0.1 M triethylammonium acetate; solvent B:CH₃CN; gradient 4-28% B in 35 min; flow rate: 1 mL/min; and detector:260 nm.

Example 3 Taqman Assay for Human Beta Actin Gene

Human genomic DNA was prepared using conventional methods. Thecomposition of the assay reagent was as follows (50 μl total volume):

Component Conc. Volume (μl) dNTPs (dATP, dCTP, dGTP, dUTP) 10 mM ea 4MgCl2 25 mM 7 ^(a)PCR Buffer, 10X — 5 UNG 1 unit/ml 0.5 Forward PCRPrimer 3 μM 5 Reverse PCR Primer 3 μM 5 AmpliTaq ™ Gold DNA Polymerase 5units/ml 0.25 Human Male DNA 10 ng/ml 2 Taqman Probe 2 μM 5 Water — 16.3^(a)10 mM KCl, 100 mM TRIS-HCl, 0.1 M EDTA, 600 nM passive internalstandard, pH 8.3.

The reagents were combined in a 96-well microtiter tray and thermallycycled using the following protocol: 50° C. for 2 min; 95° C. for 10min; 40 cycles of 95° C. for 15 sec followed by 60° C. for 1 min.Fluorescence was monitored during the amplification process using aApplied Biosystems Model 7700 Sequence Detection System (ABD).

The results of a taqman experiment can be analyzed using two parameters;the Rn value and the Ct value. The Rn value is the ratio of thefluorescence of a reporter dye and the fluorescence of a passivereference at the end of a PCR experiment. The Ct value, or thresholdcycle number, is the PCR cycle number at which the fluorescence ratio isdistinguishable from the background. For a given reporter dye and afixed concentration of target, both the Rn and Ct values reflect theefficiency of the quencher.

The efficiency of NTB 5 was compared to that of TMR in quenching thereporters FAM and TET. The Rn and Ct values for NTB and TMR wereindistinguishable for both reporter dyes. The quencher NTO 9 was usedwith FAM and found to be equivalent to both NTB and TMR. NTB was pairedwith the reporter, NED, to provide results that were similar to theother reporters. TMR could not be used as a quencher with the reporterdye NED because the fluorescence emissions of TMR and NED are at thesame wavelength.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Although only a few embodiments have been described in detail above,those having ordinary skill in the molecular biology art will clearlyunderstand that many modifications are possible in the preferredembodiment without departing from the teachings thereof. All suchmodifications are intended to be encompassed within the followingclaims.

We claim:
 1. A method for detecting a target nucleic acid sequencecomprising the steps of: providing a sample nucleic acid including atleast one target nucleic acid sequence; and hybridizing a labeledoligonucleotide probe to the target nucleic acid sequence, the labeledoligonucleotide probe being labeled with an asymmetric cyanine dyecompound having the structure:

and including any associated counter ions, wherein: n ranges from 0 to2; X is O, S or Se; R₁ is selected from the group consisting of methyland a linking group, or when taken together with the proximate carbon ofthe methine bridge forms a ring structure having from 5 to 7 members; R₂is a linking group; R₃ is hydrogen, or when taken together with R₄ formsa fused aromatic bridge substituted with one or more nitro groups; R₄ ishydrogen, or when taken together with either R₃ or R₅ forms a fusedaromatic bridge substituted with one or more nitro groups; R₅ is nitro,or when taken together with either R₄ or R₆ forms a fused aromaticbridge substituted with one or more nitro groups; and R₆ is hydrogen, orwhen taken together with either R₅ forms a fused aromatic bridgesubstituted with one or more nitro groups.
 2. The method of claim 1wherein the labeled oligonucleotide includes a reporter dye covalentlyattached to the oligonucleotide.
 3. The method of claim 2 wherein thelocation of the reporter dye and the quencher dye is such that when thelabeled oligonucleotide is hybridized to a target nucleic acid sequencethe reporter dye is not effectively quenched by the quencher dye, andwhen the labeled oligonucleotide is not hybridized to a target nucleicacid sequence the reporter dye is effectively quenched by the quencherdye.
 4. The method of claim 3 wherein when the reporter dye iseffectively quenched its fluorescence is reduced by at least a factor oftwo as compared to its fluorescence when it is not effectively quenched.5. The method of claim 4 wherein when the reporter dye is effectivelyquenched its fluorescence is reduced by at least a factor of six ascompared to its fluorescence when it is not effectively quenched.
 6. Themethod of claim 2 wherein one of the reporter and quencher dyes isattached at a 3′ end of the oligonucleotide and the other is attached ata 5′-end of the oligonucleotide.
 7. The method of claim 2 furthercomprising the step of digesting the oligonucleotide probe such that oneor both of the reporter and quencher dyes is removed from theoligonucleotide probe.
 8. The method of claim 7 wherein the step ofdigesting is effected by a 5′→3′ nuclease activity of a polymeraseenzyme.